WO2009064825A2 - Génération de nouvelles souches de vaccins bcg protégeant contre l'établissement d'infections à mycobacterium tuberculosis latentes et contre la réactivation à partir de l'état latent ou persistant - Google Patents

Génération de nouvelles souches de vaccins bcg protégeant contre l'établissement d'infections à mycobacterium tuberculosis latentes et contre la réactivation à partir de l'état latent ou persistant Download PDF

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WO2009064825A2
WO2009064825A2 PCT/US2008/083301 US2008083301W WO2009064825A2 WO 2009064825 A2 WO2009064825 A2 WO 2009064825A2 US 2008083301 W US2008083301 W US 2008083301W WO 2009064825 A2 WO2009064825 A2 WO 2009064825A2
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dosr
mycobacterium
protein
bcg
regulon
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WO2009064825A3 (fr
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Mario Alberto Flores-Valdez
Gary K. Schoolnik
Michael Klein
Jerald C. Sadoff
David Hone
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Aeras Global Tb Vaccine Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated

Definitions

  • the invention generally relates to a vaccine for treating or preventing the establishment of latent tuberculosis infections, hi particular, the invention provides a recombinant mycobacterium that overexpresses the transcription factor DosR at a level sufficient to cause induction of the dosR regulon even under non-inducing conditions.
  • Tuberculosis is a global public health problem resulting in 8 million new cases and 2 million deaths each year.
  • An estimated 2 billion persons are latently infected with Mycobacterium tuberculosis (Mtb) Reactivation of latent tuberculosis accounts for most new cases of active disease.
  • Reactivation from latent TB i.e. active tuberculosis
  • the resulting cough-generated aerosol causes dissemination of the organism to uninfected, susceptible persons, thus maintaining a transmission chain.
  • the DosR protein is thus a key regulator in the mycobacterial dormancy response.
  • Further investigations e.g. Voskuil et al., J. Exp Med. 198:705-713, 2003) demonstrated that the induction of a set of 48 genes (including dosR) adapts the Mtb organism for survival during extended periods of dormancy.
  • the 48-gene set is known as the dormancy regulon or dosR regulon.
  • Current vaccine strategies are focused on the use of the Bacillus Calmette-Guerin
  • BCG tuberculosis vaccine for the immunization of healthy persons who have never been in contact with M. tuberculosis.
  • This strategy has been of questionable efficacy and only addresses stopping active or progressive disease, without taking into account the relevance of the latent, carrier population, particularly in countries where tuberculosis is endemic. Further, the efficacy of BCG is disputed, with various controlled clinical trials and case control studies showing estimates of protection ranging from zero to 83%. A metaanalysis of the results of these trials led one group to conclude that the level of protection against pulmonary tuberculosis was 50% if the effect of latitude was included in a regression model, whereas another group concluded that these data were too heterogeneous for meaningful analysis.
  • auxotrophs e.g. for amino acids (Lee et al., Infect Immun., 74(11):6491- 6495, 2006; Sampson et al., Infect Immun. 72(5):3031-7, 2004), for nucleotide metabolism (Brown et al, 2005; Sambandamurthy et al., Nat Med.
  • reactivation is typically associated with some type of immunosuppressing condition (e.g. diabetes, aging, etc.) that allows a latent lesion to progress to active disease
  • some type of immunosuppressing condition e.g. diabetes, aging, etc.
  • No such vaccine is currently available.
  • no current vaccine, and no vaccine under development is designed to take advantage of the properties of the DosR transcriptional regulator.
  • the present invention provides a latency- or persistent-specific tuberculosis vaccine comprising a recombinant mycobacterium strain that over-expresses DosR, the latency-specific transcriptional regulator of the dormancy regulon, and thus induces the expression of the DosR regulon.
  • DosR the latency-specific transcriptional regulator of the dormancy regulon
  • dormancy regulon expression is restricted to non-replicating bacteria where cell division has been discontinued owing to environmental stimuli such as low oxygen tension or exposure to nitric oxide.
  • a genetically engineered BCG strain described herein for exemplary purposes dissociates replication from dormancy regulon expression.
  • DosR causes induction of one or more dormancy regulon genes in replicating bacteria at a level in the range of from 2 to 100 fold above that which would be observed in a non-induced, replicating mycobacterium.
  • Any genetically engineered bacterium that overproduces DosR at a level sufficient to cause a 2- 100 (or more) fold induction of dosR regulon genes or proteins may be advantageously used in the practice of this invention.
  • an immune response is elicited to the proteins encoded by the induced dosR regulon genes.
  • mycobacteria that later infect the host will be unable to establish a latent infection in the host; when the proteins required to establish a latent infection are expressed by an infecting mycobacterium, they will be recognized by the host immune system and the mycobacterium will be destroyed.
  • the ability of the mycobacteria to reactivate and progress to active disease is attenuated.
  • the latent mycobacteria may be eradicated by the immune system, or may simply be unable to emerge from the latent (persistent) state, and confined to a dormant existence. Either way, an active, contagious tuberculosis infection is prevented and the transmission to other humans is halted.
  • FIG. 1 Amino acid sequence of an exemplary DosR protein from the M. tuberculosis H37Rv dosR (devR) gene, as presented in the genome sequence available at the Pasteur Institute's TubercuList Website (SEQ ID NO: 1); B, nucleic acid sequence encoding the exemplary DosR protein of Figure IA (SEQ ID NO: 2).
  • Figure 2 Schematic representation of the integrative vector for constitutive expression of DosR in Mycobacteria.
  • Phsp ⁇ O indicates the 0.4 kb of the 5 -end of the BCG heat shock protein 60 gene locus, to which dosR is fused, and includes the promoter region, ribosome binding site, and first six codons (MAKTIA) of the hsp60 gene, as well as multiple cloning sites and the E. coli rrnABtl transcriptional terminator.
  • the Tn903 -derived aph gene (kanamycin resistance) and an origin of replication functional in E. coli (oriE) derived from pUC19 are indicated, as well as the attP sequence and int gene from the mycobacteriophage
  • the present invention provides a vaccine or immune stimulating compisitions which includes one or more genetically engineered mycobacteria that over-expresses DosR, the latency-specific transcriptional regulator (transcription factor). Persistent overexpression of DosR results in the production, by the mycobacterium, of latency associated genes and translated antigens even though the bacterium has not entered the latent phase. When compositions containing such a mycobacterium are administered as a vaccine, the vaccine recipient is therefore exposed to and mounts an immune response to the latency associated antigens being expressed by the bacterium.
  • DosR the latency-specific transcriptional regulator
  • DosR refers to the protein sequence presented in SEQ ID NO: 1 or to any other DosR protein sequence as isolated from a natural or wild-type source such as a mycobacterium.
  • DosR is also intended to encompass various variants, analogs or derivatives of such sequences if they possess an equivalent or similar ability to induce the dosR regulon.
  • Asp 54 may be replaced by glutamic acid residues so as to mimic a permanently phosphorylated (activated) DosR protein.
  • This and other site-directed mutants are also intended to be encompassed by the present invention.
  • certain non-conservative amino acid substitutions and other changes to the primary sequence may also be tolerated without disrupting the activity of DosR, and may even increase its activity.
  • non- conservative amino acid substitutions particularly in regions of the protein that are not actively involved in dosR regulon induction, may be altered.
  • various additions or deletions to the protein may be made without adverse effects, particularly short (e.g. about 25 amino acids or less) deletions or additions may be made.
  • Such changes may be desirable for any of a variety of reasons, e.g. to increase protein activity, stability, solubility, transport, etc.; to decrease susceptibility to proteases; to provide a "tag" or label (e.g. introduction of a tryptophan residue, or of a His tag sequence); etc.
  • such variants will exhibit identity with a native, wild-type DosR primary amino acid sequence in the range of about 50-60%, or 60-70%, of preferably about 70-80%, or more preferably about 80-90%, and most preferably in the range of about 90-100%.
  • Those of skill in the art are familiar with methods for the comparison of amino acid sequences and the determination of identity between or among sequences. All such variants may be utilized in the practice of the present invention, so long as sufficient ability to successfully induce the dosR regulon is retained.
  • Those of skill in the art are well-acquainted with procedures for measuring the activity of proteins, and for determining acceptable levels of activity in variant or derivative forms of proteins.
  • the activity of DosR may be measured by determining the capacity of DosR to induce expression of DosR-regulated genes under non-inducing, aerobic, conditions.
  • all DosR variants that are utilized in the practice of the present invention will retain at least about 50%, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, and most preferably at least about 90-100% (or more) of the activity of native DosR (i.e. the sequence as isolated from a natural, wild-type source, on which the variant is based).
  • the invention comprises variants of DosR that do not include the entire protein, but that include critical portions thereof, e.g.
  • variants that are utilized in the practice of the present invention will retain at least about 50%, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, and most preferably at least about
  • Figure Ib provides an exemplary nucleic acid sequence that may be used to express the DosR protein as depicted in Figure Ia.
  • nucleic acids encoding DosR is intended to include DNA, RNA, and any hybrid or combination thereof.
  • nucleic acids encoding the variants, derivatives or analogs of DosR, as described above are also encompassed by the present invention.
  • the protein is expressed at a level that exceeds that of a suitable control organism, such as the same mycobacterium that has not been genetically engineered to over-express DosR.
  • a suitable control organism such as the same mycobacterium that has not been genetically engineered to over-express DosR.
  • the over-expression of DosR in a mycobacterium may be carried out by any suitable method known in the art. Generally, the method will involve linking nucleic acid sequences encoding the DosR protein to expression control sequences that are not, in nature, linked to the dosR gene. Those of skill in the art will recognize that many such expression control sequences are known and would be suitable for use in the invention.
  • expression control sequences e.g. promoters and associated sequences
  • mycobacterium optimal promoter mop, George et al., 1995
  • blaF promoter Timm et al., 1994
  • hsp ⁇ O ace or mspl2 promoters
  • over-expression of DosR may not be constitutive but may instead be inducible, in response to an environmental cue.
  • expression of the protein may be driven by a promoter that is induced in a particular location or in response to an environmental stimulus, examples of which include but are not limited to: macrophage inducible promoter (which drives expression of genes that are specifically upregulated within the macrophage phagosome, see Schannapinger et al. JEM 2003); acetamidase promoter (Mahenthiralingam et al., J. Gen. Microbiol. 1993), and tetracycline-inducible (Blokpoel et al., Nucl. Acids Res. 33(2):e22, 2005), etc.
  • macrophage inducible promoter which drives expression of genes that are specifically upregulated within the macrophage phagosome, see Schannapinger et al. JEM 2003
  • acetamidase promoter Mcahenthiralingam et al., J. Gen. Microbiol. 1993
  • tetracycline-inducible Bookpoel et al
  • promoters from other species may be utilized, examples of which include but are not limited to: various viral promoters, whereby after “gene therapy-like” strategies (e.g. co-inoculation of mycobacteria and an engineered virus) the Mtb antigens are expressed in selected tissues infected by the co-administered virus; etc.
  • gene therapy-like strategies e.g. co-inoculation of mycobacteria and an engineered virus
  • native or naturally occurring dosR expression control sequences may be altered by genetic engineering techniques (e.g. mutations) to function in a manner that results in over-expression o ⁇ dosR.
  • nucleic acid sequences encoding the DosR protein in operable linkage with one or more expression control sequences, into a mycobacterial host where over-expression will occur.
  • the sequences may be included in a vector that is subsequently introduced into the mycobacterium.
  • vectors suitable for containing and expressing genes are known, and include but are not limited to various extra-chromosomal elements such as plasmids, e.g.
  • plasmids with origins of replication that are or will be developed; or extrachromosomal elements that do not replicate or integrate into mycobacterial genome but provide a suicidal source for homologous recombination to occur; etc.
  • Introduction of such a vector into a mycobacterium may be carried out by any of several known methods suitable for that particular vector, including but not limited to electroporation and mycobacteriophage-mediated transduction for homologous recombination.
  • any expression element may be used to house the DosR-encoding nucleic acids, and may be introduced into the mycobacterium by any suitable means, so long as the resultant genetically engineered mycobacterium expresses DosR at a level sufficient to induce the dosR regulon.
  • the vector is a plasmid and the method that is used is electroporation.
  • the DosR protein is over-expressed from the Mtb chromosome.
  • nucleic acid sequences that include one or more expression control sequences operably linked to nucleic acid sequences encoding DosR may be introduced into the bacterial chromosome, e.g. by transduction with a suicide plasmid with or without a means for counter-selection, to provide sequences for homologous recombination.
  • bovis TMC 1012 [ BCG Montreal ; CIP 105920]
  • bovis TMC 1108 [BCG Pasteur SM-R]
  • bovis TMC 1011 [BCG Pasteur], etc.
  • the recombinant mycobacteria of the invention need not be confined to strains of BCG.
  • Mycobacterium strains may also be employed, examples of which include but are not limited to: M. tuberculosis CDC1551 strain (See, e.g. Griffith et at., Am. J. Respir. Crit. Care Med. Aug;152(2):808; 1995), M. tuberculosis Beijing strain (van Soolingen et al., J CHn Microbiol. Dec:33(12):3234-8,1995) H37Rv strain (ATCC#:25618), M.
  • tuberculosis pantothenate auxotroph strain (Sambandamurthy et al., supra; M. tuberculosis rpo V mutant strain (Collins et al., Proc Natl Acad Sci USA. 92(17):8036; 1995), M. tuberculosis leucine auxotroph strain (Hondalus et al., Infect. Immun. 68(5):2888; 2000), etc., or other attenuated and/or recombinant strains derived from M. tuberculosis.
  • Other candidate bacteria include other members of the M. tuberculosis complex, other mycobacteria (e.g. M. africanum or M. avium complex bacteria), or other mycobacterial species. Any mycobacterium possessing a
  • DosR regulon with significant homology to TB complex organisms may be used in the practice of the invention, hi a preferred embodiment of the invention, the recombinant bacteria in which DosR expression is upregulated is a BCG mycobacterium, and in particular M. bovis BCG strain BCG SSI 1331.
  • the vaccine of the invention encompasses vaccines which overexpress one or more dormancy antigens, particularly dormancy antigens that are frequently recognized by otherwise healthy persons with latent tuberculosis, hi one embodiment of the invention, the antigens are selected from Rv0079, Rv0080, RvOO ⁇ l, Rv0569, RvO57O, RvO571c, RvO572c, Rv0573c, RvO574c, Rvl733c, Rvl734c, Rvl735c, Rvl736c, Rvl737c, Rvl738, Rvl812c, Rvl813c, Rvl996, Rvl997, Rvl998, Rv2003c, Rv2004c, Rv2005c,
  • antigens may include, for example, Rv0079, RvO569, Rv0572c, RvI 733c, RvI 738, Rvl813c, Rvl996, Rv2007c, Rv2029c, Rv2030c, Rv2031c, Rv2032, Rv2623, Rv2624c,
  • the vaccine of the invention may be a subunit or DNA-vaccine.
  • the vaccine would be delivered via lung pathogens.
  • the DNA sequences coding for DosR and DosR antigens could be harbored within the chromosome or extrachromosomal nucleic acid of a lung pathogen such as attenuated Pseudomonas aeruginosa, or other known attenuated fungi or viruses.
  • the nucleic acid encoding DosR and DosR antigens could be delivered by other means known to those of skill in the art, e.g. via liposomes, adenoviral vectors, etc.
  • the present invention further provides compositions for use in eliciting an immune response in and/or vaccinating a mammal.
  • the compositions may be utilized as a vaccine against Mtb, particularly against latent forms of Mtb, or forms of Mtb that are entering or emerging from latency.
  • the compositions of the invention include genetically engineered mycobacteria as described herein, alone or in combination with a pharmacologically suitable carrier.
  • the preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared.
  • the preparation may also be emulsified.
  • the active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like.
  • the composition may contain other adjuvants. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added.
  • composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration.
  • the final amount of recombinant mycobacteria in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%.
  • the compositions may further comprise additional adjuvants, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc.
  • Vaccine formulation also involves studies to determine maximum bacterial viability and stability throughout the manufacturing process. This includes determination of maximum organism viability (live to dead) during culture utilizing a variety of commonly used medium for the culture of mycobacteria to include the addition of glycerol, sugars, amino acids, and detergents or salts. After culture, cells are harvested by centrifugation or tangential flow filtration and resuspended in a stabilizing medium that allows for protection of cells during freezing or freeze-drying process. Commonly used stabilizing agents include sodium glutamate, or amino acid or amino acid derivatives, glycerol, sugars or commonly used salts.
  • the final formulation will provide sufficient viable organism to be delivered by intradermal, percutaneous injection, perfusion or oral delivery with sufficient stability to maintain and adequate shelf life for distribution and use.
  • the methods of the present invention preferably involve administering a composition comprising the recombinant mycobacteria of the invention in a pharmacologically acceptable carrier to a mammal, which is usually, but not always, a human.
  • the vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, orally, intranasally, by ingestion of a food product containing the antigen, by inhalation, etc.
  • the mode of administration is intradermal or oral, hi addition, the compositions may be administered alone or in combination with other medicaments or immunogenic compositions, e.g. as part of a multi-component vaccine. Further, administration may be a single event, or multiple booster doses may be administered at various timed intervals to augment the immune response.
  • a particular advantage of the vaccine preparation of the present invention is that while administration may be prophylactic (i.e. before exposure to the bacteria has occurred, or is suspected to have occurred) but may also be after the fact (i.e. after a known or suspected exposure) or therapeutically (i.e. after the occurrence of indicators associated with latent bacterial infection).
  • the vaccine is useful not only for preventing an initial active
  • Mtb infection but also for preventing the establishment, persistence or reactivation of a latent Mtb infection.
  • the genetically engineered mycobacterial strains of the present are tested according to methods that are well-known to those of skill in the art. For example, tests for toxicity, virulence, safety, etc. are carried out in suitable animal models, e.g. in mice, guinea pigs, etc., some of which are immunocompromised. The ability of the vaccine preparations to elicit an immune response is likewise typically tested in suitable animal models, e.g. mice, non-human primates, etc. hi addition, protection studies involving vaccination, boosting, and subsequent challenge with live Mtb may be carried out using suitable animal models such as mice, guinea pigs, and non-human primates.
  • the present invention also provides methods of eliciting an immune response to one or more of the dosR regulon proteins, which include: Rv0079, Rv0080, Rv0081, RvO569,
  • Rv0570 (nrdZ), Rv0571c, Rv0572c, RvO573c, RvO574c, Rvl733, Rvl734c, Rvl735c, Rvl736c (narX), Rvl737c ⁇ narkl), Rvl738, Rvl812c, Rvl813c, Rvl996, Rvl997 (ctpF), Rv 1998c, Rv2003c, Rv2004c, Rv2005c, Rv2006 (otsBl), Rv2007c (fdxA), Rv2028c, Rv2029c (pflcB), Rv2030c, Rv2031c (acr), Rv2032 (acg), Rv2623, Rv2624c, Rv2625c, Rv2626c, Rv2627c, Rv2628, Rv2629, Rv2630, Rv2631, Rv3126c, Rv3127, R
  • eliciting an immune response we mean that administration of the vaccine preparation of the present invention causes the synthesis of specific antibodies (at a titer in the range of 1 to 1 x 10 6 , preferably 1 x 10 3 , more preferable in the range of about 1 x 10 3 to about 1 x 10 6 , and most preferably greater than 1 x 10 6 ) and/or cellular proliferation, as measured, e.g. via cellular assays in which IFN- ⁇ production is assessed , by 3 H thymidine incorporation, etc.
  • a peptide microarray chip may be used.
  • the peptide array is spotted with overlapping 50 amino acid peptides generated from the sequences of all BCG encoded DosR-regulated proteins which are at least 2 fold induced by the overexpression of DosR.
  • Pooled sera from each group of vaccinated individuals is incubated with 3 peptide chips for 1 hr at 37 0 C.
  • the chips are then washed with phosphate-buffered saline (PBS) pH7.2 and incubated with fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG sera (Abeam, Cambridge, MA) for 1 hour at 37 0 C.
  • PBS phosphate-buffered saline
  • FITC fluorescein isothiocyanate
  • Vaccinated subjects would show higher titre responses than non-vaccinated or BCG vaccinated controls.
  • splenocytes or peripheral blood mononuclear cells (PBMCs) from vaccinated subjects are incubated with Rv2623 and Rv3130c as these proteins are known to be highly upregulated in DosR overexpressing strains and are known to be potent T cell immunogens.
  • the immune response is a protective immune response, i.e. the immune response protects the vaccinated individual from future challenge with Mtb.
  • the methods involve administering a composition comprising a mycobacterial strain of the present invention in a pharmacologically acceptable/compatible carrier.
  • the dosR regulon proteins that are produced are those that are most highly induced, that elicit a strong IFN- ⁇ response, that are potent T cell or B cell immunogens, and possess high sequence homology to their Mtb homologue.
  • this would likely include bacterial components that are readily accessible to cells of the immune system (e.g. surface exposed) that elicit such IFN- ⁇ release with no effect upon production of anti-inflammatory molecules.
  • the dosR regulon genes that are expressed or proteins which are transcribed in response to DosR induction are produced at a level that is in the range of from about 2 to about 100 fold greater than the level that is observed in a mycobacterium in which DosR is not over-expressed, e.g. a control or wild type aerobically replicating mycobacterium.
  • this overproduction of gene products, as measured by transcription, or antigens translated from the transcribed genes, is what is meant by induction.
  • the fold increase is from about 2-10, or 10-20, or 20-30, or 30-40, or 40-50, or 50-60, or 60-70, or 70-80, or 80-90, or 90-100, or even more than a 100 fold increase is observed.
  • at least one of the 48 dosR regulon genes is produced at this level, and preferably from about 1- 5, or 5-10, or 10-15, or 15-20, or 20-25, or 25-30, or 30-35, or 35-40, or 40-45, or up to all
  • a recombinant strain of the invention may be used as a tool to specifically diagnose people latently infected with Mycobacterium tuberculosis
  • recombinant BCG overexpressing DosR-antigens are cultivated under non-inducing conditions to prepare whole cell extracts, and the IFN- ⁇ , cell-mediated response is compared to that of wild type BCG or Mtb to distinguish between people with active and latent Mtb infection.
  • EXAMPLE 1 Construction of a BCG strain constitutively expressing the latency-specific transcriptional regulator, dosR
  • DosR also known as DevR
  • Rv3133c a vector was constructed in which dosR was expressed under the control of the hsp ⁇ O promoter.
  • the wild-type M. tuberculosis dosR gene was amplified from Mtb strain 1254 using the primers devR-FPvu2 (5 GTGC AGCTGTC ATGGT AAAG GTCTTCTTGGTCG-3, SEQ ID NO: 3) and devREHd3 (5 ' -ACTAAGCTTCCTGTTGTC ATGGT CCATC ACCG-3, SEQ ID NO: 4).
  • the devR-FPvu2 primer was designed so that it maintains the correct Open Reading Frame (ORF) in the vector, under the control of the /?s/?60-promoter, and so that it includes a change in the start codon of dosR from GTG to ATG.
  • ORF Open Reading Frame
  • the 216 amino acids residues encodied by the PCR product are identical to those encoded by the M. tuberculosis H37Rv dosR (devR) gene, as presented in the genome sequence available at the TubercuList Website located at genolist.pasteur.fr.
  • the PCR reaction was performed using Platinum Pfx High Fidelity Supermix (Invitrogen) and genomic DNA from wildtype M. tuberculosis 1254 as a template.
  • the resulting PCR products were cloned into a pCR4 vector plasmid, using a TOPO cloning system (Invitrogen).
  • the resulting plasmid is depicted schematically in Figure 2.
  • selection of recombinant clones was accomplished by growing colonies on Luria broth (LB) plates containing 50 ⁇ g/mL of kanamycin. Plasmid DNA from selected clones was isolated and sequenced, and one plasmid containing an insert with a sequence 100% identical to that of wild type M.
  • tuberculosis H37Rv dosR was selected for further work.
  • the genetic makeup of pMF361dosR was confirmed by restriction digestion and sequencing.
  • M. bovis BCG SSI 1331 cells were transformed with plasmid pMF361dosR by electroporation, and electroporated cells were plated onto 7H10 media plates supplemented with OADC (Difco) and 25 ⁇ g/mL of kanamycin. After 4 weeks of growth, cells from individual clonal colonies were selected and grown in liquid 7H9 media supplemented with ADC (Difco) plus 25 ⁇ g/mL of kanamycin. After culturing for 3 weeks in this media, genomic DNA from the selected clones was isolated and checked for the integration of pMF361dosR into the BCG genomic attB site.
  • M. tuberculosis strains that constitutively express DosR (Mtb 1254: :DosR, H37Rv: :DosR, and
  • H37Rv ⁇ Rv3132c-Rv3134c::DosR strains constitutive production of DosR by the recombinant strain (as measured by the capacity of DosR to induce expression of DosR-regulated genes under non-inducing, aerobic, conditions) was documented by comparing the transcriptional profiles of BCG SSI1331::DosR and wild type BCG SSI1331 grown in vitro. The strains were assayed for dosR induction at early, mid-log and stationary phase during growth in 7H9 ADC 0.05% Tween 80 liquid media, at 37°C, 100 rpm, and 5% CO 2 .
  • DosR-OX whereas BCG cells carrying only plasmid pMV361 (lanes labeled “vector”) did not induce DosR-regulated genes, when compared to wild type expression.
  • hi vitro dosR is induced either by growing M. tuberculosis under hypoxic conditions or exposing the microorganism to nitric oxide treatment (Voskuil et al., supra); ex vivo, dosR is activated during macrophage infection (Schnappinger et al., supra).
  • Table 1 (lane labeled "H37Rv dormancy"). The number of DosR binding sites present (in strain Park MM03) is given in the last column.
  • the numbers indicate the fold-change in the expression of a particular gene in comparison to the strain containing only pMV361 (vector control).
  • all the samples correspond to cultures at the corresponding OD 600 nm as reference.
  • the reference is mid- log phase aerobic culture as reported by Voskuil et al., supra. Numbers in bold indicate genes for which expression in the recombinant strain was higher than that observed in the reference strain.
  • *ND indicates a value that was not significantly different between the recombinant strain and the reference. **NR stands for "not reported”.
  • the numbers indicate the fold-change in the expression of a particular gene in comparison to the strain containing neither pMV361 nor pMF361dosR. For these experiments , all the samples correspond to cultures at the corresponding OD 600 run as reference.
  • This example demonstrates that stable and constitutive expression of DosR was obtained under non-inducing conditions, and that such constitutively produced DosR was able to activate genes of the DosR-regulon despite the presence or absence of the chromosomally encoded copy of this gene.
  • a constitutive gain-of- function mutation is sought by mutating the DosR active site Asp (Asp54) to GIu.
  • GIu is isosteric with phosphorylated Asp and thus the protein carrying this mutation may behave as though it is phosphorylated.
  • This strategy has proven to be effective for at least three response regulators: OmpR, NtrC and RcsB.
  • the active site constitutive mutant is produced as a single copy gene driven by the hsp ⁇ O promoter and integrated (together with a selectable antibiotic or other marker) into the chromosome at the attB site. This prevents loss of the gene, a possible consequence complicating the expression of the constitutive mutant from a multicopy, episomal, plasmid.
  • the recipient strain that is used is a deletion mutant of wild type DosR to facilitate evaluation of the mutant and to prevent competition between the wild type DosR protein and the mutant protein for the same promoter sequence or possible dominant-negative effects that might occur if both wild type and mutant protein interact in the cytosol.
  • a minimum of three variants of the same stain are assessed for expression of the dormancy regulon under two in vitro conditions of growth.
  • Condition 1 hi well-stirred (i.e., oxygenated) mid-log phase cultures in 7H9 medium where prior studies have shown the dormancy regulon to be silent. Under this condition, the DosR regulon is silent in the BCG wild type strain and in the dosR deletion mutant, but expressed in the dosR deletion mutant complemented by the activating dosR mutation.
  • Condition 2 hi the Wayne low O 2 model of latency (day 10) where prior studies of
  • the DosR regulon is expressed by the BCG wild type strain and by the dosR deletion mutant complemented by the activating dosR mutation.
  • the dormancy regulon is silent in the dosR deletion mutant.
  • the MTB oligonucleotide microarray is used to assess expression of the BCG dormancy regulon because the nucleotide sequences of 25 of the 48 BCG genes are absolutely conserved with the corresponding H37Rv MTB gene; 15 have a single SNP; five have 2 SNPs; and, two have 3 SNPs when compared to the sequenced MTB strain from which the microarray was derived, hi each case, the gene expression profile is compared using two MTB reference RNAs: RNA from MTB strain H37Rv aerated mid log phase cultures in 7H9 medium (this reference represents basal levels of dormancy regulon expression); and RNA from H37Rv low oxygen Wayne model cultures (this reference represents maximal levels of dormancy regulon expression).
  • mice The capacity of the parental BCG and engineered strain to elicit a cellular immune response is compared to dormancy regulon protein in mice.
  • BCG-DosR To illustrate the immune responses elicited by BCG-DosR and to demonstrate its protective efficacy against tuberculosis, four groups of 10 C57/BL6 mice are vaccinated subcutaneously with either 1) saline, 2) BCG, 3) BCG-DosR. Mice in groups 2, 3, and 4 receive 5xlO 5 cfu of BCG or recombinant BCG. After 10 weeks, five mice from each group are sacrificed for immune assays and the remaining animals are challenged with 100 cfu aerosolized M. tuberculosis Erdman.
  • a peptide microarray chip is utilized. The chip is spotted with overlapping 50 amino acid peptides generated from the sequences of all BCG encoded DosR-regulated proteins which are at least 2 fold induced by the overexpression of DosR. Pooled sera from each group is incubated with 3 peptide chips for 1 hr at 37 0 C. The chips are then washed with phosphate-buffered saline (PBS) pH7.2 and incubated with FITC conjugated goat anti-mouse IgG sera (Abeam, Cambridge, MA) for 1 hour at 37 0 C.
  • PBS phosphate-buffered saline
  • mice receiving BCG-DosR show greater antibody responses to peptides derived from DosR regulated proteins than unvaccinated mice or mice vaccinated with BCG or BCG-PfoA.
  • spleens are harvested from each and homogenized. Splenocyte concentrations are adjusted to 5x10 5 cells/well in
  • mice from each group are sacrificed 10 weeks after challenge with M. tuberculosis and lungs and spleens are aseptically harvested from each animal. Lungs and spleens are homogenized in PBS and dilutions are plated on 7H10 agar. After 4 weeks of incubation, the number of M. tuberculosis colonies are enumerated for each animals lungs and spleen and corrected for the dilution factor. This value is the number of live tubercle bacilli present in the lungs and spleen of each animal.
  • BCG vaccination typically results in approximately a 1 log reduction in cfu/lung and spleen versus saline vaccination.
  • BCG-DosR vaccination results in a greater reduction of Mtb load in the lungs and spleens of mice.

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Abstract

L'invention concerne un vaccin destiné au traitement ou à la prévention de l'établissement d'infections tuberculeuses latentes. Le vaccin comprend une mycobactérie recombinante qui surexprime le facteur de transcription DosR à un niveau suffisant pour induire la production des gènes ou des protéines du régulon DosR. Un hôte recevant le vaccin monte une réponse immunitaire aux protéines du régulon DosR et est ainsi protégé contre l'établissement, la persistance ou la réactivation d'une tuberculose latente.
PCT/US2008/083301 2007-11-13 2008-11-13 Génération de nouvelles souches de vaccins bcg protégeant contre l'établissement d'infections à mycobacterium tuberculosis latentes et contre la réactivation à partir de l'état latent ou persistant WO2009064825A2 (fr)

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WO2015104380A1 (fr) 2014-01-09 2015-07-16 Transgene Sa Fusion d'antigènes mycobactériens hétérooligomères
US9211529B2 (en) 2009-09-11 2015-12-15 E I Du Pont De Nemours And Company Conversion of ethanol to a reaction product comprising 1-butanol using hydroxyapatite catalysts

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EP3360566B1 (fr) 2009-11-20 2019-12-25 Oregon Health&Science University Procedes destines a detecter une infection par mycrobacterium tuberculosis
TWI458978B (zh) * 2011-12-27 2014-11-01 Chengchung Chou 判別開放性或潛伏性結核菌感染之方法
CN113481232A (zh) * 2021-07-13 2021-10-08 潍坊医学院 结核分枝杆菌DosR缺失菌株的构建及检测DosR调控蛋白生理功能及作用的方法
CN115028695B (zh) * 2022-05-24 2024-05-17 中国人民解放军总医院第八医学中心 基于LTBI-RD相关蛋白的Th1和CTL表位肽池及其应用
CN114736276B (zh) * 2022-05-24 2023-08-01 中国人民解放军总医院第八医学中心 结核分枝杆菌ltbi-rd相关蛋白抗原的ctl表位肽及其应用

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US9211529B2 (en) 2009-09-11 2015-12-15 E I Du Pont De Nemours And Company Conversion of ethanol to a reaction product comprising 1-butanol using hydroxyapatite catalysts
WO2015104380A1 (fr) 2014-01-09 2015-07-16 Transgene Sa Fusion d'antigènes mycobactériens hétérooligomères
US10765731B2 (en) 2014-01-09 2020-09-08 Transgene Sa Fusion of heterooligomeric mycobacterial antigens

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